Activating Autoantibodies against the Calcium-Sensing Receptor Detected in Two Patients with Autoimmune Polyendocrine Syndrome Type 1

Abstract

Context: Autoimmune polyendocrine syndrome type 1 (APS1) is an autosomal recessive disorder caused by mutations in the autoimmune regulator (AIRE) gene. Hypoparathyroidism occurs in 80% of patients with APS1 and has been suggested to result from an autoimmune reaction against the calcium-sensing receptor (CaSR) in parathyroid cells. Anti-CaSR binding antibodies have previously been detected in patients with APS1.

Objective: The aim of this study was to determine whether anti-CaSR antibodies present in APS1 patients could modulate the response of the CaSR to stimulation by Ca²⁺.

Results: The results indicated that two of the 14 APS1 patients included in the study had anti-CaSR antibodies that stimulated the receptor. These antibodies were detected by their ability to increase both Ca²⁺-dependent extracellular signal-regulated kinase phosphorylation and inositol phosphate accumulation in human embryonic kidney 293 cells expressing the CaSR.

Conclusion: An important implication of the present results is that although the majority of APS1 patients do not have CaSR-stimulating antibodies, there may be a small but substantial minority of patients in whom the hypoparathyroid state is the result of functional suppression of the parathyroid glands rather than their irreversible destruction.

The presence of calcium-sensing receptor stimulating antibodies in patients with autoimmune polyendocrine syndrome type 1 is discussed.

Autoimmune polyendocrine syndrome type 1 (APS1) is a rare autosomal recessive disorder (1) caused by mutations in the autoimmune regulator (AIRE) gene (2,3), which encodes a putative transcription factor (4,5). The disease, also known as autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), is characterized by multiple organ-specific autoimmunity and ectodermal manifestations (1,6). In the majority of cases, disease components include mucocutaneous candidiasis, hypoparathyroidism, and Addison’s disease, with type 1 diabetes mellitus, alopecia, vitiligo, autoimmune hepatitis, and pernicious anemia occurring less frequently (1,6). APS1 patients typically display a wide variety of antibodies against enzymes found in the affected organs, including 17-α-hydroxylase (7) 21-hydroxylase (8,9) and side-chain cleavage enzyme (9), all of which are present in the adrenal cortex. Antibodies against pancreatic glutamic acid decarboxylase 65 and tyrosine phosphatase-like protein IA-2, which are prevalent in autoimmune type 1 diabetes mellitus, can also be detected in individuals with APS1 (10,11). Other identified autoantigens include tryptophan hydroxylase (12), tyrosine hydroxylase (13), and aromatic l-amino acid decarboxylase (14), which are present in APS1 patients with intestinal dysfunction, alopecia, and autoimmune hepatitis, respectively. In addition, the transcription factors SOX9 and SOX10 are vitiligo-associated autoantigens in APS1 (15). More recently, interferon antibodies have been identified in almost all patients with this condition, making them a diagnostic marker for APECED (16,17).

Hypoparathyroidism occurs in 80% of patients with APS1 and is associated with hypocalcemia, hyperphosphatemia, and low serum levels of PTH. Early reports suggested that the clinical symptoms and biochemical manifestations of hypoparathyroidism could result from humoral immune responses to parathyroid cells (18,19,20). Subsequently, parathyroid autoantigens targeted by autoantibodies in APS1 patients have been identified as the NACHT leucine-rich-repeat protein 5 (NALP5) (21) and the G protein-coupled calcium-sensing receptor (CaSR) (22,23,24,25), which plays a pivotal role in maintaining calcium homeostasis by sensing circulating calcium levels and regulating PTH synthesis and release as well as renal calcium excretion (26).

Recently, we confirmed the presence of anti-CaSR antibodies in a cohort of patients with APS1 using a novel immunoprecipitation assay (24). The aim of the current study was to identify anti-CaSR antibodies in APECED patients that affect the activity of the CaSR, because antibody-induced activation of the receptor could result in low levels of PTH and thus contribute to the clinical picture of hypoparathyroidism in APS1. The effects of patient IgG on the function of the CaSR were assessed by measuring their effects on both Ca²⁺-dependent extracellular signal-regulated kinase (ERK1/2) phosphorylation and inositol phosphate (IP) accumulation in human embryonic kidney 293 (HEK293) cells expressing the CaSR.

Patients and Methods

Patients and controls

Fourteen patients (seven males, seven females; mean age, 18 yr; range, 10–47 yr) were studied. All patients had Addison’s disease and mucocutaneous candidiasis. Thirteen patients had hypoparathyroidism. Other autoimmune diseases present were: premature ovarian failure, five; alopecia, three; vitiligo, five; type 1 diabetes mellitus, three; autoimmune hypothyroidism, one; and pernicious anemia, two. All patients carried mutations in both alleles of the AIRE gene (3). In our previous study, anti-CaSR antibodies were detected in 12 of these patients using immunoprecipitation assays, whereas the remaining two were negative (24).

Twenty healthy individuals (nine males, 11 females; mean age, 32 yr; range, 24–48 yr) who had no present or past history of autoimmune disorders were the control subjects. No individual had anti-CaSR antibodies when tested in immunoprecipitation assays (24).

The study was approved by the Ethics Committee of Tampere University Hospital, Tampere, Finland, and the APS1 patients participated in the study after obtaining informed consent, given either by the patient or by their parents. Sera were stored at −20 C.

IgG preparation

IgG was isolated from sera by affinity column chromatography using protein G-Sepharose 4 Fast Flow (Amersham Biosciences AB, Uppsala, Sweden) according to the manufacturer’s instructions. Eluted IgG fractions were extensively dialyzed against PBS (pH 7.4; Sigma, Poole, UK) and concentrated using an Amicon Concentrator (Amicon Inc., Beverly, MA). IgG samples were sterilized with a Millex Filter Unit (Millipore Corp., Bedford, MA), and the final concentrations were measured by photometry at 280 nm. All IgG samples were stored at 10 mg/ml at −20 C.

Cell culture

HEK293 cells and HEK293 cells stably expressing the CaSR (HEK293-CaSR) (27) were cultured in Dulbecco’s DMEM medium (Invitrogen, Paisley, UK) containing 4.5 g/liter pyruvate, 10% (vol/vol) fetal calf serum, 100 U/ml penicillin G, 100 μg/ml streptomycin, 2 mm l-glutamine (all from Invitrogen) at 37 C in a 95% humidified atmosphere of 5% CO₂. Transfections of HEK293 cells using a calcium phosphate transfection system (Invitrogen) were carried out exactly as previously detailed (24). Plasmid pDEST26/CaSR, containing human CaSR cDNA, was obtained from RZPD (German Resource Centre for Genome Research, Berlin, Germany). Plasmid pcMCHR1 carrying human melanin-concentrating hormone receptor (MCHR1) cDNA is described elsewhere (28).

Cell extracts were prepared from untransfected and transfected HEK293 cells as detailed elsewhere (24). Total protein content of cell extracts was determined using a Bio-Rad Protein Assay Kit (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK). Extracts were diluted where appropriate to contain equivalent amounts of total protein and stored at −80 C until required.

Measurement of CaSR-stimulated inositol-1-phosphate (IP1) accumulation

The response of HEK293-CaSR cells to Ca²⁺ was assessed by measuring intracellular IP1 accumulation (27). HEK293-CaSR cells were plated in 24-well plates at 4 × 10⁵ cells per well in 500 μl of culture medium and incubated overnight at 37 C. Cell monolayers were washed first with serum-free medium and then with buffer containing 146 mm NaCl, 2.5 mm KCl, 10 mm LiCl₂, 0.2 mm MgCl₂, 2.8 mm glucose, and 10 mm HEPES (pH 7.4). Cells were subsequently incubated for 60 min at 37 C in 200 μl of buffer alone or containing varying concentrations of Ca²⁺ (as CaCl₂). After treatment, cells were lysed for 30 min at 37 C with 50 μl of 2.5% IP-One ELISA Kit Lysis Reagent (CIS Bio International, Gif-sur-Yvette, France). The accumulation of intracellular IP1 was measured according to an IP-One ELISA Kit (CIS Bio International), an immunoassay based on competition between free IP1 and IP1-horseradish peroxidase (HRP) conjugate for binding to anti-IP1 monoclonal antibody. The results for IP1 accumulation were expressed as: percentage inhibition of IP1-HRP binding = [1 − IP1-HRP binding in stimulated cells/IP1-HRP binding in unstimulated cells] × 100. An increase in IP1 accumulation in the HEK293-CaSR cells is reflected by an increase in the percentage inhibition of IP1-HRP binding.

For measuring the effects of APS1 patient and control IgG on CaSR-stimulated IP1 accumulation, HEK293-CaSR cell monolayers were incubated with IgG at a 1:20 dilution in 100 μl of buffer for 10 min at 37 C. Cells were then incubated for 60 min at 37 C with a further 100 μl of buffer alone or buffer containing Ca²⁺ before measuring IP1 accumulation. Each experiment included HEK293-CaSR cells stimulated with Ca²⁺ alone and HEK293-CaSR cells left untreated with IgG and Ca²⁺. For dilution experiments, IgG samples were tested at dilutions of 1:50, 1:100, 1:200, 1:500, and 1:1000 with Ca²⁺. To determine the effects of heat treatment, IgG samples were treated at 100 C for 10 min before testing at a dilution of 1:20.

For absorption experiments, IgG samples were incubated with either untransfected or transfected (with pDEST26/CaSR or pcMCHR1) HEK293 cell extract (300 μg of total protein) at 4 C for 16 h in buffer containing 150 mm NaCl, 10 mm Tris-HCl (pH 7.4), 1% (vol/vol) Triton X-100 and Protease Inhibitor Cocktail (Sigma). IgG samples without cell extract were incubated under the same conditions. After absorption, samples were centrifuged at 45,000 × g for 1 h at 4 C, and the supernatants were recovered. For effects upon CaSR-stimulated IP1 accumulation, IgG samples were tested at a 1:20 dilution.

Measurement of CaSR-stimulated ERK1/2 phosphorylation

The response of HEK293-CaSR cells to Ca²⁺ was assessed by measuring the phosphorylation of ERK1/2 (27). HEK293-CaSR cells were plated in 96-well plates at 1.5 × 10⁴ cells per well in 100 μl of culture medium and incubated overnight at 37 C. Cells were then incubated with serum-free medium overnight. Cell monolayers were washed first with serum-free medium and then with buffer. Cells were subsequently incubated for 60 min at 37 C in 100 μl of buffer alone or buffer containing varying concentrations of Ca²⁺. After treatment, cells were fixed for 20 min at room temperature with 100 μl of 4% Cell Fixing Buffer according to a Cellular Activation of Signaling ELISA (CASE) Kit (SuperArray Bioscience Corporation, Frederick, MD). The phosphorylation of ERK1/2 was then measured according to a CASE Kit (SuperArray Bioscience Corporation), and the results were expressed as: ratio of phosphorylated ERK1/2:total ERK1/2.

For measuring the effects of APS1 patient and control IgG on CaSR-stimulated ERK1/2 phosphorylation, HEK293-CaSR cell monolayers were incubated with IgG at a 1:20 dilution in 100 μl of buffer for 10 min at 37 C. Cells were then incubated for 60 min at 37 C with a further 100 μl of buffer alone or buffer containing Ca²⁺ before measuring ERK1/2 phosphorylation. Each experiment included HEK293-CaSR cells stimulated with Ca²⁺ alone and HEK293-CaSR cells left untreated with IgG and Ca²⁺.

Statistical analyses

Statistical analyses were performed using unpaired Student’s t tests. The prevalence of antibody reactivity to the CaSR was compared between APS1 patients and controls using Fisher’s exact test for 2 × 2 contingency tables. In all tests, P values <0.05 (two-tailed) were regarded as significant.

Results

Effects of APS1 patient IgG on CaSR-stimulated IP1 accumulation

The response to Ca²⁺ of the CaSR expressed in HEK293 cells was assessed by measuring intracellular IP1 accumulation (27) as described in Patients and Methods. To determine the effects of IgG from APS1 patients (n = 14) and healthy controls (n = 20) on CaSR function, HEK293-CaSR cells were incubated with IgG samples at a 1:20 dilution before no stimulation or stimulation with various concentrations of Ca²⁺ (0.5, 1.5, 3.0, and 5.0 mm). Each experiment included HEK293-CaSR cells stimulated with Ca²⁺ alone and HEK293-CaSR cells left untreated with IgG and Ca²⁺. IP accumulation was measured using the IP-One ELISA Kit as detailed in Patients and Methods, and the percentage inhibition of IP1-HRP binding was calculated.

Of the 20 control IgG samples analyzed, none had any effect upon the levels of IP1 accumulation when compared with stimulation by Ca²⁺ alone (Fig. 1). Similar results were obtained for 12 of the 14 APS1 patient IgG samples tested (Fig. 1). In contrast, IgG samples from two APS1 patients (APS1-2 and APS1-15) significantly increased the levels of IP1 accumulation at Ca²⁺ concentrations of 0.5, 1.5, and 3.0 mm when compared with Ca²⁺ stimulation alone (P values <0.0001) (Fig. 1). The IgG from these two patients had no effect on IP1 accumulation, however, when no Ca²⁺ was added to the buffer, indicating that some degree of receptor activation was needed to see the functional effect of the IgG. The results indicated the presence of CaSR-stimulating activity in these two APS1 patient IgG samples. Both of these patients were positive for anti-CaSR binding antibodies (24) and had hypoparathyroidism as part of their APS1.

Figure 1.

Effect of APS1 patient and control IgG on the response of the CaSR to Ca²⁺ stimulation by measuring IP1 accumulation. Changes in IP1 accumulation were measured in response to no Ca²⁺ and Ca²⁺ at 0.5, 1.5, 3.0, and 5.0 mm in HEK293-CaSR cells preincubated with either APS1 patient (n = 14) or control (n = 20) IgG samples at a 1:20 dilution. IP accumulation was measured in the IP-One ELISA Kit as detailed in Patients and Methods. The results show the mean percentage inhibition of IP1-HRP binding (±sd) in three experiments in which HEK293-CaSR cells were preincubated with IgG from APS1 patients 2, 3, 4, 5, controls 1, 2, 3, 4, 5, and no IgG (A); IgG from APS1 patients 2, 6, 7, 8, 9, controls 6, 7, 8, 9, 10, and no IgG (B); IgG from APS1 patients 2, 10, 11,12, 13, controls 11, 12, 13, 14, 15, and no IgG (C); and IgG from APS1 patients 2, 14, 15, controls 16, 17, 18, 19, 20, and no IgG (D).

To determine the effects of diluting IgG on CaSR-stimulated IP1 accumulation, HEK293-CaSR cells were incubated with IgG samples from APS1 patients APS1-2 and APS1-15 (both with CaSR-stimulating activity) and from two controls at dilutions of 1:20, 1:50, 1:100, 1:200, 1:500, and 1:1000 before stimulation with Ca²⁺ at 1.5 mm. The limiting dilutions for the detection of CaSR-stimulating activity in IgG samples from APS1 patients APS1-2 and APS1-15 was 1:200 (Fig. 2): only IgG dilutions of up to 1/200 were noted to significantly increase the levels of IP1 accumulation when compared with Ca²⁺ stimulation alone (P values <0.05) (Fig. 2).

Figure 2.

Effect of diluting APS1 patient and control IgG on the response of the CaSR to Ca²⁺ stimulation. Changes in IP1 accumulation were measured in response to Ca²⁺ at 1.5 mm in HEK293-CaSR cells preincubated with IgG samples at dilutions of 1:20, 1:50, 1:100, 1:200; 1:500, and 1:1000. IP accumulation was measured in the IP-One ELISA Kit as detailed in Patients and Methods. The results show the mean percentage inhibition of IP1-HRP binding (±sd) of three experiments for IgG from APS1 patients APS1-2 and APS1-15 and from two controls.

To determine the effects of heat treatment of the IgG from APS1 patients APS1-2 and APS1-15 and from two controls on CaSR-stimulated IP1 accumulation, HEK293-CaSR cells were incubated with IgG samples (heat treated and untreated) at a dilution of 1:20 before stimulation with Ca²⁺ at 1.5 mm. Heat treatment of IgG samples from APS1 patients APS1-2 and APS1-15 before testing significantly reduced the CaSR-stimulating effects of the samples on HEK293-CaSR cells compared with untreated IgG (P < 0.05) (Fig. 3). This result indicates that the effects of the IgG samples on HEK293-CaSR cells were not simply due to contaminating Ca²⁺ or some other nonspecific, heat-stable factor.

Figure 3.

Effects of heat treating APS1 patient and control IgG on the response of the CaSR to Ca²⁺ stimulation. Changes in IP1 accumulation were measured in response to Ca²⁺ at 1.5 mm in HEK293-CaSR cells preincubated with IgG samples at a dilution of 1:20. Both heat-treated and untreated IgG samples were tested. IP accumulation was measured in the IP-One ELISA Kit as detailed in Patients and Methods. The results show the mean percentage inhibition of IP1-HRP binding (±sd) of three experiments for heat-treated and untreated IgG from APS1 patients APS1-2 and APS1-15 and from two controls.

To confirm the specificity of the CaSR-stimulating activity in the two IgG samples, absorption experiments were undertaken. Samples of IgG from APS1 patients APS1-2 and APS1-15 and from two controls were preabsorbed with extract from either HEK293 cells or HEK293 cells expressing the CaSR or the MCHR1 resulting from transfection with either pDEST26/CaSR or pcMCHR1, respectively. Preabsorbed and unabsorbed IgG samples were then incubated at a 1:20 dilution with HEK293-CaSR cells before stimulation with 1.5 mm Ca²⁺. Preabsorption of IgG from APS1 patients APS1-2 and APS1-15 with extract from either HEK293 cells or HEK293 cells expressing MCHR1 did not significantly decrease the stimulatory effect of the samples upon HEK293-CaSR cells compared with unabsorbed IgG samples (P > 0.05) (Fig. 4). In contrast, the stimulatory effect upon HEK293-CaSR cells was significantly reduced in both APS1 patient IgG samples preabsorbed with HEK293 cells expressing the CaSR compared with unabsorbed IgG samples (P < 0.05) (Fig. 4). The absorption experiments indicated that the stimulatory activity present in the IgG samples from both APS1 patients reflected the presence of antibodies that specifically interacted with the CaSR.

Figure 4.

Effect of preabsorbing APS1 patient and control IgG on the response of the CaSR to Ca²⁺ stimulation. Changes in IP1 accumulation were measured in response to Ca²⁺ at 1.5 mm in HEK293-CaSR cells preincubated with IgG samples at a dilution of 1:20. IgG samples had been preabsorbed by cell extracts of either HEK293 cells or HEK293 cells transfected with either pDEST26/CaSR or pcMCHR1. Unabsorbed IgG samples were also tested. IP accumulation was measured in the IP-One ELISA Kit as detailed in Patients and Methods. The results show the mean percentage inhibition of IP1-HRP binding (±sd) of three experiments for IgG from APS1 patients APS1-2 and APS1-15 and from two controls.

Effect of APS1 patient IgG on CaSR-stimulated ERK1/2 phosphorylation

The response to Ca²⁺ of the CaSR expressed in HEK293 cells was assessed by measuring ERK1/2 phosphorylation (27) as detailed in Patients and Methods. To determine the effects of IgG from APS1 patients APS1-2 and APS1-15 on CaSR function with respect to ERK1/2 phosphorylation, HEK293-CaSR cells were incubated with IgG samples at a 1:20 dilution before no stimulation or stimulation with various concentrations of Ca²⁺ (0.5, 1.5, 3.0, and 5.0 mm). Each experiment included HEK293-CaSR cells stimulated with Ca²⁺ alone and HEK293-CaSR cells left untreated with either IgG or Ca²⁺. The phosphorylation of ERK1/2 was measured according to a CASE Kit as in Patients and Methods, and the ratio of phosphorylated ERK1/2:total ERK1/2 was determined. None of the two controls was observed to have an effect upon the levels of ERK1/2 phosphorylation when compared with Ca²⁺ stimulation alone (Fig. 5). In contrast, IgG samples from the two APS1 patients (APS1-2 and APS1-15) were noted to significantly increase ERK1/2 phosphorylation at Ca²⁺ concentrations of 0.5 and 1.5 mm when compared with Ca²⁺ stimulation alone (P values <0.05) (Fig. 5). As with the stimulatory effect of IgG from these two patients on IP1 accumulation, no effect of the IgG was observed in the presence of nominal zero Ca²⁺. The results indicated the presence of CaSR-stimulating activity in IgG samples from the two APS1 patients.

Figure 5.

Effect of APS1 patient and control IgG on the response of the CaSR to Ca²⁺ stimulation by measuring ERK1/2 phosphorylation. Changes in ERK1/2 phosphorylation were measured in response to no Ca²⁺ and Ca²⁺ at 0.5, 1.5, 3.0, and 5.0 mm in HEK293-CaSR cells preincubated with either APS1 patient (n = 2) or control (n = 2) IgG samples at a 1:20 dilution. The phosphorylation of ERK1/2 was measured according to a CASE Kit as in Patients and Methods. The results show the mean ratio of ERK1/2 phosphorylation (±sd) of three experiments for preincubation of HEK293-CaSR cells with IgG from APS1 patients APS1-2 and APS1-15, IgG from two controls and no IgG.

Discussion

Previously, we identified anti-CaSR antibodies in patients with APS1 using a novel immunoprecipitation assay (24) supporting the view that the CaSR can be a target for autoantibodies in APECED patients (22,23,25). The aim of the present study was to determine whether anti-CaSR antibodies in APS1 patients were able to modulate the function of the CaSR because such antibodies are more likely to have a direct role in disease pathogenesis than antibodies that simply bind the receptor. Indeed, in several autoimmune disorders, antibodies have been demonstrated to affect the functioning of cell receptors resulting in disease manifestations. For example, thyroid-stimulating antibodies activate the TSH receptor and elicit hyperthyroidism in Graves’ disease by mimicking the binding of TSH, and antibodies directed against the acetylcholine receptor block the acetylcholine-binding site and provoke accelerated receptor degradation causing myasthenia gravis (29,30). In the case of the CaSR, antibody-induced activation could result in low levels of PTH and the resultant hypoparathyroidism that is associated with APS1.

Overall, our results indicated that two of 13 (14%) of the hypoparathyroid APS1 patient IgG samples tested exhibited readily detectable and statistically significant activation of the CaSR. Although such antibodies were not found at a significantly increased prevalence in the APS1 patient group when compared with healthy controls in our comparatively small sample size (P = 0.16), nonetheless these results are compatible with the appearance of stimulating anti-CaSR antibodies in a subset of patients. The anti-CaSR binding antibodies previously demonstrated in the remaining 10 APS1 patients (24) may bind at a site on the receptor that has no effect upon functionality. An analogy might be with the detection of stimulatory antibodies in only a subset of the entire patient population with TSH receptor autoantibodies (29). Alternatively, CaSR-modulating activities in these samples may be too low to detect in the assays used in this study.

Of interest, the two APS1 patient IgG samples that stimulated IP1 accumulation and ERK1/2 activity did so only in the presence of Ca²⁺ in the experimental buffer. This observation is similar to that seen with the so-called type 2, calcimimetic CaSR activators (31). The latter require the presence of physiological levels of extracellular Ca²⁺ to activate the CaSR. In the cases of both the activating IgG studied here and the calcimimetics, binding of antibody or calcimimetic may act in an allosteric manner to favor the active conformation of the receptor, but Ca²⁺ must be present to initiate productive signal transduction.

In addition to APS1, anti-CaSR antibodies have been demonstrated in individuals affected by isolated autoimmune hypoparathyroidism (AH) (22,23,27,32) and autoimmune hypocalciuric hypercalcemia (AHH) (33,34,35). With respect to CaSR function, anti-CaSR activating and blocking antibodies have only been detected previously in two patients with AH and five patients with AHH, respectively (27,33,34). In addition, a unique anti-CaSR antibody from an individual with AHH has been reported to both stimulate the Ca²⁺-dependent accumulation of inositol phosphates and inhibit the Ca²⁺-dependent phosphorylation of ERK1/2 (35). The anti-CaSR antibodies detected in APS1 patients in this study were similar in their effects upon Ca²⁺-dependent receptor activity to those identified earlier in individuals with AH (27): IgG samples increased both Ca²⁺-dependent IP accumulation and ERK1/2 phosphorylation in HEK293 cells expressing the receptor.

An important implication of the present results is that although the majority of APS1 patients do not have CaSR-stimulating antibodies, there may be a small but substantial minority of patients in whom the hypoparathyroid state is the result of functional suppression of the parathyroid glands rather than their irreversible destruction. The documentation of the inactivating anti-CaSR antibodies in AHH proves that the presence of anti-CaSR antibodies need not produce destruction of the parathyroid glands (33). In addition, our earlier demonstration of the presence of activating antibodies to the CaSR in one patient with transient hypoparathyroidism as well as in a second hypoparathyroid patient with a morphologically intact parathyroid gland despite many years of hypoparathyroidism proves that activating antibodies also need not irreversibly destroy the parathyroids (27). In such cases, it is possible that a calcilytic CaSR inhibitor might be of diagnostic and/or therapeutic utility. Because such agents acutely stimulate PTH secretion by blocking the inhibitory effect of calcium on the CaSR, demonstrating a PTH response in a hypoparathyroid subject administered a calcilytic could document the presence of residual functioning parathyroid tissue. Moreover, if such functioning tissue were present, the calcilytic might be of therapeutic utility if it were able to block the suppressive effect of the antibody on parathyroid function.

To summarize, the present study is the first to define specific anti-CaSR antibodies in patients with APS1 that simulate the receptor, albeit in only two of 13 such patients with hypoparathyroidism, and also provides further evidence for the CaSR as a humoral immune target in APECED.